J. Mol. Biol. (1992) 226, 491-505

Protein Interactions

with Urea and Guanidinium

Chloride

A Calorimetric Study George I. Makhatadze and Peter L. Privalovt Department

of Biology

The Johns Hopkins University, Baltimore, MD 21218, U.S.A. and the Institute of Protein Research of the Academy of Sciences of Russia Puschino, Russia (Received 14 August

1991; accepted 24 March

1992)

The interaction of urea and guanidinium chloride with proteins has been studied solutions with denaturants at various fixed calorimetrically by titrating protein temperatures, and by scanning them with temperature at various fixed concentrations of denaturants. It has been shown that the observed heat effects can be described in terms of a simple binding model with independent and similar binding sites. Using the calorimetric data, the number of apparent binding sites for urea and guanidinium chloride have been estimated for three proteins in their unfolded and native states (ribonuclease A, hen egg white lysozyme and cytochrome c). The intrinsic and total thermodynamic characteristics of their binding (the binding constant, the Gibbs energy, enthalpy, entropy and heat capacity effect of binding) have also been determined. It is found that the binding of urea and guanidinium chloride by protein is accompanied by a significant decrease of enthalpy and entropy. At all concentrations of denaturants the enthalpy term slightly dominates the entropy term in the Gibbs energy function. Correlation analysis of the number of binding sites and structural characteristics of these proteins suggests that the binding sites for urea and guanidinium chloride are likely to be formed by several hydrogen bonding groups. This type of binding of the denaturant molecules should lead to a significant restriction of conformational freedom within the polypeptide chain. This raises a doubt as to whether a polypeptide chain in concentrated solutions of denaturants can be considered as a standard of a random coil conformation.

Keywords: proteins;

denaturants;

denaturation;

Urea and guanidinium chloride (GdmCl$) are widely used as protein denaturants. Nevertheless, notwithstanding the extensive literature on protein denaturation by these agents, the mechanism of this reaction is still obscure. We are not even certain whether the action of these agents on proteins is direct and can be regarded as ligand binding, or if it is indirect and involves a change in the properties of solvent (water) in the presence of urea and GdmC1 (Schellman, 1978, 1987a,b; Arakawa & Timasheff, 1984; Breslow & Guo, 1990; Creighton, 1991; Hedwig et al., 1991). The poor understanding of this process is due, primarily, to a lack of direct experi-

t Author to whom all correspondence should be addressed. $ Abbreviations used: GdmCl, guanidinium chloride; Rns. bovine pancreatic ribonuclease A; Lys, hen egg white iysozyme: Cyt, rytochrome c; a-Cyt, apo-Cyt.

491 $03.00/O

calorimetry

mental information on urea and GdmCl interaction with proteins. Most of the available information, except the earlier studies of Tanford on the transfer of amino acid residues into aqueous solution of denaturants (for a review, see Tanford, 1970) and that of Gill et al. (1961) and Robinson & Jencks (1965) on the solubilities of model compounds in these solutions, is based on indirect observations of changes in protein conformation in the presence of denaturant (see, e.g. Tanford, 1968; Pace, 1975, 1986, 1990). This allows one to specify the relative stability of the native protein state using various extrapolations, but cannot provide the details of the mechanism of protein interaction with denaturants that are needed for understanding the physical basis of the energetics of the native protein structure. To go into these details, one needs, first of all, to know the number of moles of denaturant bound to protein. But how does one measure the number of moles of urea or GdmCl bound to protein. when the concentration of protein is 10e4 M and the concentration of denaturant is 6 M (Schellman, 1987612

1. Introduction

0022-2836/92/140491-15

thermodynamics;

0 1992 Academic Press Limited

492

G. I. Makhatadze and P. L. Privalov

One of the approaches to this experimental problem consists of measuring the volume effects accompanying apparent binding of urea and GdmCl to protein (Lee & Timasheff, 1974; Prakash et al., 1981). However, the volume effects of binding are too small for a detailed thermodynamic analysis of this process, which requires variation of a conjugate intensive parameter, i.e. pressure. From this point of view, the study of the heat effects of denaturants’ interaction with protein is much more promising. This is not only because the heat effect of denaturant binding is larger than the volume effect, and can be measured with higher accuracy using modern microcalorimetric techniques, but because the conjugate intensive parameter of the heat of the reaction is temperature. Variation of temperature with measurement of the corresponding heat effect opens a prospect for a detailed thermodynamic analysis of the phenomenon of ligand binding (Wyman & Gill, 1990). It is surprising, therefore, that calorimetry has not been widely used to solve interaction of protein with the problem denaturants. After the first quantitative calorimetric study of lysozyme denaturation by GdmCl (Pfeil & Privalov, 19763), which demonstrated the efficiency of this method in development of protein thermodynamics, almost nothing has been done in this direction. The short communication by Pfeil et al. (1991), on the calorimetric study of GdmCl interaction with the unfolded polypeptide chain, has appeared only recently. In this paper we show that using isothermal reaction and scanning microcalorimetry one can get a complete set of data required for the thermodynamic description of the denaturant interaction with proteins. This has been done on three globular proteins: pancreatic ribonuclease A, egg white lysozyme and cytochrome c. These proteins were chosen because their three-dimensional structure is well known, which is important for correlating the thermodynamic and structural information, and because their denaturation process and their denatured states have been extensively studied by various physico-chemical methods, particularly in our laboratory. It has been shown that ribonuclease and lysozyme with disrupted disulfide crosslinks and the apo-form of cytochrome are completely unfolded in solution at acidic pH (Privalov et al., 1989; Privalov t Makhatadze, 1990). This is important for studying the denaturant interaction with a polypeptide chain because, in the completely unfolded state, all potential binding sites are exposed to the solvent and their number does not of the denaturant variation upon change concentration.

2. Materials and Methods Bovine pancreatic ribonuclease A (Rns), hen egg white lysozyme (Lys) and their reduced forms (Rns-” and Lys-““) with disrupted disulfide and sulfhydryl residues blocked by carboxymethylation, together with bovine heart cytochrome c (Cyt) and its apo form (a-Cyt) were

Table 1 Dependence of the activity of urea and GdmCE on the molarity of solution at different temperatures Activity GdmClg

Urea3 Molarityt 1 2 3 4 5 6 7 8

10°C

25°C

40°C

10°C

25°C

40°C

0946 1.823 2.656 3469 4281 5113 5,980 6899

@964 1%80 2772 3661 4564 5495 6-467 7.488

0.984 1.931 2.868 3818 4796 5809 6859 7.940

0293 0.818 1.436 2.234 3342 4852 6724 ~

0305 0887 1.608 2.565 3918 5792 8.174

0.312 0936 1.741 2831 4392 6580 9.373 -.

t At 25°C.

$ Data taken from Stokes (1967). QData taken from Makhatadze

et al. (unpublished

results).

obtained, purified and tested for purity as described in our previous paper (Privalov e6 al., 1989). This paper also provides a description of all the procedures of solution preparation for the calorimetric experiments and protein concentration measurements. Most experiments were done in the presence of 30 mM-&Cine buffer (pH 25 to 30) or acetate buffer (pH 30 to 50) without salts. The urea used in our experiments was analytical grade (U.S.S.R.). Guanidinium chloride (GdmCl) was purchased from Merck (Germany). The concentrations of urea and GdmCl in solution were determined refractometrically using tabulated values of the solution refractive index (Pace, 1986). The activity of urea in solution with given molality (M) in the temperature range 10 to 40°C was taken from Stokes (1967). The activity of GdmCl in this temperature range was determined by measuring the heat effects of dilution at the corresponding temperatures (G. I. Makhatadze, E. Freire, J. Fernandez, T. H. Lilley & P. L. Privalov, unpublished results). The activity values of

GdmCl and urea at 10, 25 and 40°C are listed m Table 1. Protein solution was prepared by dissolving lyophilized protein in the solvent of given activity followed by careful dialysis against this solvent. Therefore, the activity of denaturant in protein solution was very close to that in the corresponding solvent without protein. Heat values of isothermal protein reaction with denaturants were measured in the flow microcalorimeter LKB 10700-l as described (Pfeil & Privalov, 1976a,b). Because the heat values of dilution of urea and GdmCl are too large, the calorimetric titration was done stepwise by changing the molarity of reagent by no more than one unit at each step and integrating the stepwise results. At each step the base line was obtained by recording the heat of mixing the solvent with the reagent concentration (cl) and the flow rate (vi), and the solvent with the reagent concentration (c2) and the flow rate (vJ. The final concentration of reagent after mixing will be c, = (c, w1 +c2v2)/

(q +z+). Then the solvent with the reagent concentration c1 is replaced by the protein solution, which was dialyzed against this solvent. The observed shift of calorimetric recording from the base Iine just corresponds to the heat effect of protein interaction with the reagent upon changing centration

the reagent concentration c, to final cr.

The heat of temperature-induced and the partial

heat capacities

from

the initial

con-

protein denaturation

of protein

in the considered

Protein Interaction

Table 2

F. Heat effect

(Ml

- QNt

-Q”t

-QNt

-Q”t

-QNt

-Q”t

1.0 90 30 40 50 60 7.0 8.0

706 123-I 1757 212.5

1208 2402 3436 4463 5192 587.3 651.0 728.0

50.8 103.2 1544 19893 248.4 309.9 358.4 406.3

1006 193.3 2862 369.0 4362 5059 5654 6281

43.9 83.2 113.9 137.9 1643 181.2 200.9 2159

88.2 1697 2399 304.3 370.0 422.8 484.0 5381

@) 1.0 20 30 40 50 6.0 7.0 C. Heat

-QN

-Q”

1195 2092 307.7 4151 510.8 5750 611.3

193.9 3450 460.3 5581 636.4 701.4 7895

t&at&in

by GdmCl

at variow

25°C

115.8 195.9 266.0 317.5 379.0 466.2 510.5

e#ect of ribonuclease

e&N

151.7 3003 419.1 510.9 5907 6663 7397

titration

10°C

107.3 1848 232.2

by urea

1352 2639 3938 4w7 549.4 6253 7150 at various

25°C

@)

-QN

-Q”

1.o 2.0 30 40 50 6.0 7.0 8.0

60.2 113.8 167.4 2184 278.1

114.5 223.3 312.8 39%6 467.0 5359 5948 653.0

D. Heat effect temperatures

GdmCl (M) 1.o 2.0 30 40 50 64 7.0 E. Heat

of

ribonuclease

-QN

46.4 88-4 120.4 153.0 185.1 232.7 250.0 321.2

-Q”

-Q’J

-QN

90.3 176.2 254.8 327.3 3952 4557 517.3 567.2

27.9 438 438 33.7 494 81.6 1034 1234

171.7 351.4 457.6 571.8 6366 691.0 7394

w&N

72.8 1392 202.6 267.2 321.2 38@6 4209 4681

40°C -Q”

78.1 1454 212.4 262.8 3340 4008 450.2

-Qu

by GdmCi at various

25°C -Q”

89.1 182.1 274.2 355.4 436.2 4895 544.8

40°C

titration

10°C

m&N

1302 260 1 4040 501.8 5669 6356 7043

effect of eytochrome c titration

-Qu

43.8 696

1157 223.4 331.7 433.7 512.7 578.5 637.4

by urea at various

temperatures

Urea (M) 1.0 2.0 30 40 50 60 7.0 84

10°C -QN

25°C -Q”

108.1 196.6 281.5 356.1 4168 458.6 491.7 532.8

-QN

30.5 55.0 77.7 loo-2 130-6 140-2 171.7 210.7

1.0 2.0 3.0 40 50 6.0 7.0

-Q”

e&N

-Q”

1107 216.0 322.2 381.9 4495 511.6 5850

836 134.9 1748 2327 2968 347.4 3764

99.7 200.3 310.4 3756 452.0 521.0 5745

t @ and Q”, kJ mol-‘,

-QN

-Q”

863 1922 2854 3731 441.1 497.7 548.4

throughout.

solutions were measured with a DASM-4 scanning microcalorimeter, as described (Privalov & Potekhin, 1986).

40°C -Q”

81.1 159.1 221.1 2853 346.6 4060 451.3 5097

m&N

3. Results

-Qu

temperatures

Urea

-QN

40°C -Q”

-QN

(q

4oO”c

25°C

40°C

25°C

B. Heat effect of lysozyme temperatures 10°C GdmCl

10°C

GdmCl

of lysozyme titration by uw.a at various temperatures 10°C

of cytochrome c titration by GdmCl at various

temperatures

Heat effects of titration A. Heat efled Urea

493

with Denaturants

-Qu

61.7 122.0 177.1 2273 269.2 302.4 3424 3768

(a) Isothermal calorimetric titration Figure 1 and Table 2 represent the results of calorimetric titration by GdmCl and urea at 25°C of the completely unfolded polypeptide chains of three proteins: Rns?, Lys?” with disrupted disulfide bonds and the apo form of Cyt. As shown (Privalov et al., 1989), the polypeptide chains of these proteins in acidic solutions (30 mM-glycine, pH 2.5) are in a conformation that resembles closely a random coil. The Table gives the molar titration enthalpies calculated per mol of protein molecule; the Figure shows the specific heat effects, calculated per gram of protein, plotted as functions of denaturant activities. The specific heat effects of titration of the unfolded polypeptide chains are very similar for all three proteins studied, but the titration curves for GdmCl and urea differ significantly. The similarity of the specific heat effects of different polypeptide chains titrated by the same reagents is not surprising since the amino acid content of t,hese globular proteins is rather similar. The titration curves, especially in the case of urea, are far from saturation at the practical limit of the denaturant concentration (8 M). Nevertheless, the heat effect of titration at this concentration is quite large, about 50 J g-l protein, i.e. several hundred kJ mol-‘, for both reagents. It should be noted that the similar value of the total heat effect of protein interaction with GdmCl at its limited concentration was obtained by Pfeil et al. (1991) for apo-cytochrome c. However, according to these authors the titration curve is somewhat steeper and much closer to saturation in 7 M-solution than that found in our experiments. The reason for this deviation in the shape of the titration

curves

is unclear.

The calorimetric titration curves for the native proteins differ significantly between themselves, in contrast to those of the unfolded proteins. Each of these curves is very specific for the given protein and its shape depends on the pH of solution (Fig. 1, filled symbols). This should be expected, since, in

G. I. Makhutadze and P. L. Privalov

494

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2

3

4

5

6

7

8

90

I

2

3

4

5

6

7

Activity (a)

(b)

Figure 1. Specific heats of protein titration by (a) GdmCl and (b) urea at 25°C. Filled symbols correspond to the initially native proteins, open symbols correspond to the initially unfolded proteins. (0) Rns+’ pH 2.5; (0) Rns pH 5.0 in GdmCl and pH 2-5, 3.5 in urea; (IJ) Lys-“’ pH 2.5; (m) Lys pH 2.5 (a) and (b); (A) a-Cyt pH 2.5, (A) Cyt pH 52 (b).

the case of native protein, the titration heat effect includes the heat effect of protein denaturation induced by the increasing concentration of denaturant. Therefore, the final heat effect of titration of the unfolded and native protein should differ by the enthalpy of protein unfolding. A comparison of this difference of heat effect on titration of the native and unfolded proteins with the enthalpy of protein thermal denaturation at the same temperature in the absence of denaturant, which was determined by scanning microcalori-

0

metry (Privalov & Khechinashvili, 1974), shows that they are in a perfect correspondence: for Rns both in GdmCl and urea it is 19( + 1) Jgg’ and for Lys it is 15( + 1) Jg-’ at 25°C. For Cyt, a direct comparison is impossible because of the heme that is absent from the apo-form. The correspondence of the enthalpy of protein temperature-induced denaturation, determined by with the scanning microcalorimetric studies, enthalpy of protein unfolding determined from the titration curves, demonstrates the accuracy of our

I

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I

w

2

4

6

8

IO

o123456789 Act&y

(al

Figure 2. Specific heats of titration and 40°C.

I

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I

(b)

of the unfolded Rns by (a) GdmCl and (b) urea at 3 different temperatures:

10, 25

Protein Interaction

495

with Denaturants

0.4

i 0, o-3 T x

3

D

2

0.2

0-I

I I

0

I ii

I 3

I 5

I 4

I 6

I 7

I 0

I 2

I I

I 3

I 5

I 4

I 6

I 8

I 7

9

Molarlty (a)

(b)

Figure 3. Partial specific heat capacity increment of unfolded polypeptide (a) GdmCl and (b) urea. The symbols are the same as for Fig. 1. calorimetric results. This is important because the titration of protein by stepwise calorimetric denaturants is not a simple procedure experimentally and might involve considerable errors accumulated in the consecutive steps. It also confirms our previous conclusion that the denatured states of protein obtained by heat; acid or GdmCl denaturation are indistinguishable enthalpically (Pfeil & Privalov, 19766; Privalov, 1979; Privalov et al., 1989). Figure 2 presents the results of calorimetric titration of RnspSS with GdmCl and urea at three fixed temperatures: 10, 25 and 40°C. Similar results have been obtained for all other proteins studied. Corresponding experimental data are given in Table 2. As seen from the Figure and Table, the heat effect of protein titration by GdmCl and urea depends significantly on temperature: with decreasing temperature, the slope of the titration curves becomes steeper and the final value of the heat effect at 8 M-denaturant increases. This is just what one could expect if the enthalpy of denaturant binding by protein were negative. Comparing the values of the titration heat at the same denaturant concentration but a different temperatures, one can determine the partial heat capacity

increment

of protein

chains of proteins caused by the presence of

the denaturant concentration increasing asymptotically with its increase. For GdmCl, the asymptotic value of the heat capacity increment is reached at the concentration of about 4 M and amounts to 62 to 63 J K-’ gg’ protein. For urea the asymptotic value is approached only at the concentration of 8 M and appears to be much larger, about 045 J K-’ g-l protein. (b) Scanning microcalorimetry Figure 4 represents the heat effects that are observed by the scanning calorimeter upon heating protein solutions in the presence of denaturants. With an increase of the denaturant concentration the protein thermostability decreases and it denatures at lower temperatures. The decrease of thermostability is accompanied by a decrease in the

Table 3 Partial speci$ic heat capacity increment of unfolded polypeptide chains of proteins caused by the presence of GdmCE and urea in aqueous solutions at 25°C

caused by denaturant

SC’, (J K-’

solvation:

g-l)

GdmCl

b(J = &(*2)--&v,) ‘P

T,-T,



where Q(T) is the specific heat of protein titration at temperature T. The corresponding results for the mean temperature (25°C) are listed in Table 3 and shown in Figure 3. The errors in determining these derivative functions are not small, but the heat capacity effects of binding are quite considerable, both for GdmCl and urea. They clearly depend on

Urea

M

cyt,

RNase

Lyz

Cyt

RNaM!

Lyz

1-o 2.0 3-O 40 50 60 7.0 8-o

0.07 @07 @ll 0.03 0.02 0.04 0.10 ~

0.14 0.31 @31 @34 0.30 028 0.25

0.14 019 016 0.21 0.20 0.18 0.17

0.13 0.21 0.30 @37 042 045 0.43 0.44

0.10 0.21 027 0.32 0.36 0.38 0.43 0.45

0.08 0.16 0.24 033 @35 038 0.39 044

G. I. Makhatadze and P. L. Privalov

496

OM

70

1~n

60 i

i

Y

50

E E 3

40

:: om b

30

20

IO

I IO

I 20

I 30

I 40

I 50

I 60

I 70

I 80

I I I I 90 0 IO 20 Temperature (“C)

dependence of the partial specific heat capacity various concentrations of (a) GdmCl and (b) urea.

denaturation heat effect, judging by the decrease in the heat absorption peak area. The decrease of the denaturation heat effect means a decrease of the enthalpy and entropy of denaturation (see Table 4 and Fig. 5). The presence of GdmCl and urea leads to a noticeable increase of the partial heat capacity of proteins, especially in the unfolded state (Fig. 4). The partial specific heat capacity of the unfolded polypeptide chain of RnsPS at 25°C is 190( +O*Ol) J K-’ g- ‘, but in the presence of 3 M-urea it increases to 2.10( +O*Ol) J K-’ g-‘, i.e. by 620 J KP1 gg’. This

I 40

I 50

I 60

I 70

I 80

I 90

I( 0

(b)

(a)

Figure 4. Temperature containing

I 30

of Rns in aqueous solutions at pH 55

just what is expected from the calorimetric titration studies (see Table 3). Using the found values of SC, one can easily calculate the partial heat capacity of protein in solution without denaturant from the partial heat containing capacity of protein in solution The partial specific heat capacity denaturant. values of protein corrected for the solvation effect are in agreement, within experimental error, with the values obtained directly for the proteins in solution without denaturant (Fig. 6). This supports our earlier conclusion that the apparent difference

600

Temperature PC) (a)

Figure 5. Temperature dependence of the enthalpy of heat denaturation containing various concentrations of (a) GdmCl and (b) urea.

(b)

of Rns (0)

and Lys (0)

in solutions

Protein Interaction

497

with Denaturants

Figure 6. Partial specific heat capacity of unfolded (a) a-Cyt and (b) Rns-SS in solutions containing various concentration of urea. The pH of all solutions was 2.5. Open circles correspond to the values corrected for denaturant salvation effect.

in heat capacities of denatured proteins in the presence and absence of denaturant is mainly caused by the denaturant solvation effect and not by the difference in protein conformation (Pfeil & Privalov, 19766). It should be noted that our results on the heat capacity effects of urea interaction with proteins do not confirm earlier calorimetric studies of Kresheck & Benjamin (1964), according to which this heat capacity effect is negative.

4. Discussion (a) Analysis of binding

isotherms

Since the mechanism of protein interaction with urea and GdmCl is still a subject of hot discussion, we can start its consideration from the simplest assumption that it simply represents binding of denaturant molecules to specific binding sites of the protein (Schellman, 1955; Tanford, 1970). This is an alternative representation to the linear free energy model (Schellman, 1987a,b, 1990; Pace, 1986, 1990) and it will be interesting to see whether this simple binding model is able to describe quantitatively all the heat effects that we observe experimentally. If we assume independent binding sites for a number of denaturant molecules (X) bound by the protein molecule in solution with denaturant activity a, then:

X(a) = hi 1 I,x ; a’ I where ni is the number of i-type binding sites and k, is their binding constant. If Ahi is the enthalpy of binding of denaturant by the i-type site, then the heat effect of protein titration by the denaturant is: (3) Since we do not know anything definitely the protein-binding sites for urea or GdmCl,

about we can

498

G. I. Makhatadze and P. L. Privalov 400

300

7 0 E 3

200

c$

100 ”

3--n, ”

I 100

0

I

I

200

I

300

400

I

I

500

600 -0

(a) Figure 7. Scatchard plots of the calorimetric

titration

x

s.

Under n we assume here an effective number of equivalent binding sites and under k an effective binding constant. Upon rearrangement, we get Scatchard’s expression, which is widely used for treating titration data (see e.g. Wyman & Gill, 1990): &(~)/a

= @Ah) x k-k

x Q(a).

loo

I

I

300

400

(J mol-‘1

(5)

A plot of Q(a)/a against Q(o) should give a straight line if the binding sites are independent and similar. The slope of this line corresponds to the binding constant k and the abscissa intercept gives (nAh) if Q is calculated per mol of protein or (nAh)/M if Q is calculated per gram of protein. The analysis of the calorimetrically measured heat effects of titration of the unfolded polypeptide chains by GdmCl and urea show that their Scatchard plots are close to linear (Fig. 7). Thus, the calorimetric titration results are likely to be described, within experimental errors, by the simple model suggested above, where the binding of GdmCl and urea molecules occurs at independent and similar binding sites on the protein. However, since the errors in the &(a)/~ values increase with decreasing activity (Wyman & Gill, 1990), we preferred to determine the binding parameters in equation (5) by analyzing titration curves using the method of non-linear regression with a commercial PC program Enzyme Fitter (Biosoft, Cambridge, U.K.). This method does not require a large extrapolation and gives more reliable results,

I

I

500

600

(b)

data at 25°C for apo-cytochrome

suppose, in the first approximation, that they are similar and are not distinguished by the binding constants and thermodynamic characteristics of binding. This simplifies equation (3):

Q(a) = nAh

I

200

I

0

;:

c in

(a)

GdmCl and (b) urea.

especially at low activities of denaturants. The values of k and (nAh) obtained according to the simple binding model for the three unfolded polypeptide chains at three different temperatures are given in Table 5. As one can expect from the similarity of corresponding titration curves, the binding constant for these polypeptide chains are very similar, but for GdmCl binding they differ considerably from the value 1.16 found by Pfeil et al. (1991) in their recent calorimetric experiments with apocytochrome c, which showed a somewhat steeper titration curve than that observed in this work. From the binding constant. and its temperature dependence we can estimate the Gibbs energy of binding: Ag=-RTlnk, the enthalpy

(6)

of binding: Ah

=

4&/T) -zzz

RT2

d

and the entropy

In

Ic

(7)

dT’

41/T)

of binding: d In k

As=--$f=Rlnk+RTF.

When (nAh) and Ah are determined, mate the number of binding sites: n

-

(nAh)

Ah

.

(8)

we can esti-

(9)

All the int.rinsic thermodynamic characteristics of urea and GdmCl binding to the individual binding sites of protein are listed in Table 5. The obtained value of the binding constant for urea to protein is higher than that obtained for binding of urea to peptides by isopiestic vapor pressure measurements, 6038 (Schonert & Stroth,

Protein Interaction

499

with Denaturants

Table 5 Thermodynamic parameters

of GdmCl and urea binding to proteins Denaturant GdmCl

Urea Thermodynamic parameters k for: C‘ytochrome c Ribonuclease Lpsozyme kt Ah (kJ molF I) at 25°C for: C’,vtochrome e Ribonuclease Lysozgme Ah? (kJ mol-‘) Ah. n for: (‘ytochrome c Ribonuclease Lysozyme Agt (kJ mol-‘) Aat (J K- 1 mol - ‘) nt for: C’ytochrome c Ribonuclease I,ysozymr t i\vrraged

10°C

25°C

40°C

10°C

25°C

0.097 @068 @070 0.078 +0016

0071 0.052 0.059 0.061 +0.010

0068 0.049 0.049 0.055 +0011

0.67 0.84 039 0.80 kO.12

0.51 0.60 0.69 04.50 + 0.09

-9 -8 -9 -9&2 - 1360 - 2060 -2210

-1390 - 2030 - 2030 6.9+1 -53&-8 142*19 240 + 28 229* 16

042 0.53 061

0.51 + 0.09

-12 -11 -10 -11*2 - 1080 -1660 - 1930

- 660 - 870 - 860

- 690 - 820 - 820 1.3* 1 -4lfX

-680 -740 -770

56k2 74&8 82k6

from values for all 3 proteins

1981), and for binding of urea to diketopiperazine by solubility measurements of this compound in urea solutions, 0.0423 and 0049 (Sijpkes et al., 1992). The enthalpy value found of urea binding to protein is very close to the value obtained by Robinson & Jenks (1965) from the solubility studies of the compounds modeling peptides in urea solutions (- 11.7 kJ mol-‘) but is somewhat smaller than that’ obtained by Gill et aE. (1961) from the solubilit’y studies diketopiperazine (- 142 kJ mall’) and by SEionert 8: Stroth (1981) (- 18.7 kJ mol. ‘). (b) Intrinsic

40°C

binding characteristics

Analysis of the intrinsic binding characteristics shows both differences and similarities between the interaction of urea and GdmCl with proteins. They differ, first of all, in the binding constants. The binding constant for urea is an order of magnitude lower than that for GdmCl. This is evident even from a comparison of the corresponding titration curves (Fig. 1): in 8 M-urea they are much further from saturation than in 7 M-GdmCl. On the other hand, the enthalpies of binding of these two compounds to polypeptide chains do not differ much. Therefore, the difference in the binding constants is mainly caused by the entropies of binding. The entropies of binding are negative, large in magnitude and significantly larger for urea than for GdmCl. Most remarkable is that the negative entropy of binding overcompensates the effect of

enthalpy of binding, so that the Gibbs energy of binding is positive. The other distinguishing feature of GdmCl and urea binding is that nAh values are different in these two cases and, correspondingly, the numbers of binding sites (n) for urea and GdmCl in the unfolded polypeptide chain are different. Taking into account the values of binding constants of GdmCl (0.600) and urea (6061) at room temperature, we find, by the simplified equation (2), that the unfolded polypeptide chain of RnssSS which has 74 binding sites for GdmCl and 240 sites for urea, in 6 M-solutions should bind about 57 molecules of GdmCl and 60 molecules of urea. For Lys~“” which has 82 binding sites for GdmCl and 229 sites for urea, we get 64 bound molecules of GdmCl and 58 molecules of urea. Therefore, the number of GdmCl and urea molecules actually bound by the polypeptide chains is not too large and not too different from these two compounds, notwithstanding very large differences in their binding constants. It is interesting to compare these numbers of bound GdmCl and urea molecules with those obtained by other authors. According to densitometric studies of Lee & Timasheff (1974) and Prakash et al. (1981), in 6 iw-GdmCl, the Rns molecule binds 72 and Lys 67 molecules of GdmCl; in 6 M-urea, Rns binds 57 and Lys 65 molecules of urea. The close correspondence of these data with those obtained by us, using a quite different approach, is impressive.

500

G. I. Makhatadze and P. L. Privalov Table 6 Binding parameters for the unfoldins process at 25°C A&X at various concentrations of denaturant at 25°C (M)

Denaturant

GdmCl Urea

Protein CYt RNase LYZ CYt RNase LYZ

t A&X* was calculated

QN/Q”

n"

nN

nU-nN

1

2

3

4

5

6

0.66 058 0.69 0.32 0.51 0.52

56 74 82 142 240 229

37 43 57 48 122 119

19 31 25 94 118 110

2.9 48 39 52 66 6.1

6.6 10.8 87 9.7 12.1 11.3

9.3 152 12.3 136 17.1 15.9

11.5 18.8 152 17.2 21.5 201

133 21.7 17.5 205 257 240

148 24.1 194 236 29.6 27.6

from the effect of 1 M-denaturant

on the transition

Comparing the heat effect of titration of the unfolded polypeptide chain (Q”) with that of the native protein (QN) at the denaturant concentration that certainly does not unfold native protein, we can determine the number of binding sites for urea and GdmCl in the native protein nN: nN = (QN/QU) x n”.

(10) Knowing nN and n” we can estimate the number of ligands that are bound to the protein upon its unfolding: A$Y(a) = (n”-nN)

ka x ~ l+ka’

temperature

A&Y*?

3.9 51 3.0 2.9

according to eqn (20).

In contast to the intrinsic Gibbs energy of binding, it is a negative quantity that increases in magnitude with the increase of the denaturant activity in solution. Therefore, protein unfolding, which is associated with the increase of the number of binding sites, is a thermodynamically favorable process and is promoted by the increasing activity of denaturant. For the contribution of each binding site into the total entropy and enthalpy change of protein caused we have bY the presence of denaturant correspondingly: dA6 A&(a) = - dT = Rln(l+ka)+

These numbers for the proteins investigated in GdmCl and urea solutions at various concentrations are given in Table 6.

d In k dT + s

RTz(a) (

(c) Total thermodynamic eflects of binding Perhaps most surprising in the intrinsic binding characteristics obtained of GdmCl and urea is that the Gibbs energies of their binding are positive, i.e. the binding is unfavorable thermodynamically. This immediately raises the question: if the binding of these substances is unfavorable, why do they unfold the compact native protein structure by increasing the number of the binding sites? The answer is obvious. All the proteins considered in this work are single-domain proteins, and they each act as a single co-operative system. The change of macroscopic state of such a system depends on the total change of its thermodynamic characteristics. In the case of protein unfolding it depends on the total change of the Gibbs energy upon unfolding. Taking into account the combinatorial effects of realization of the macroscopic state by many microscopic states (see Schellman, 1955, 1978; Tanford, 1968; Wyman & Gill, 1999), the total Gibbs energy change caused by all possible bindings in the n sites is:

A&(,)

= _ ‘;@$$’

If the activity’s ignored:

, >

(14)

= RT2 d ‘“11’T’ ka)

temperature

dependence can be

AS^(a) = AsT?(a) - R In kff(a) + R ln(1 + ka), A&(a) = AL%!(a).

(16) (17)

(12) Therefore, the contribution of each of the binding sites into the total Gibbs energy change of protein caused by the presence of denaturant is:

Thus, the total enthalpy change is just proportional to the extent of binding by the single site (see eqn (4)). However, the entropy effect has a much more complex expression: the two additional terms to the intrinsic entropy term in equation (16) are actually reflecting the combinatorial effect in the realization of the considered macroscopic state by the number of microscopic states. Most important is that both these terms are positive. Therefore, they decrease the negative intrinsic entropy effect of binding, i.e. the effect of fixation of denaturant molecules on the protein. Since the enthalpy of denaturant binding by a single binding site is:

A&a) = - RT ln( 1 + ka).

Afi(a) = AH(a)/n = Q(a)/n,

AG(a) = -nRT

ln( 1 + ka).

(13)

(18)

Protein Interaction

501

with Den&wants

Table 7 The number of bound molecules of urea and GdmCl and corresponding enthalpy, entropy and Gibbs energy effects per single binding site Urea Molarity 1 2 3 4 5 6 7 8

AI? (kJ mol-‘)

d 0056 0103 0145 0183 0.218 0.251 0283 0314

AL? (J K-’

CdmCI mol-‘)

-1.2 -2.1 -31 -37 -4.7 -53 -56 -6.3

- 0.5 -@9 - 1.3 -1.6 -2.0 -2.3 - 2.5 -28

A6 (J mol-‘) -140 -270 -390 -500 -610 - 720 - 820 -930

where &(a) is the calorimetrically measured heat of protein titration, the entropy of denaturant binding per single binding site can also be determined as: A,.‘?(a)= [AA(a) - A@(a)]/T =TnQ(a) +Rln(l+ka).

(19)

The total thermodynamic effects of binding of GdmCl and urea to proteins, normalized to a single binding site, are presented in Table 7. (d) An alternative approach to the bonding phenomenon The number of denaturant molecules that are bound to a protein upon its unfolding can also be found from the depression of protein transition temperature, T,, in the presence of denaturant (see Fig. 5 and Table 4). The number of bound ligands in this case can be found (see Privalov et al., 1969; Schellman, 1975, 1976) as:

A”X* = &$Wt) N

RT2

dT,

ri

At? (kJ mol-‘)

This equation is valid when the concentration of ligands in solution is significantly lower than the concentration of the main solvent, in our case, water (Tanford, 1970). The numbers of AgX*, which we get for 1 M-GdmCl and urea solutions by applying equation (14) to the data given in Table 3, are listed in the last column of Table 6. A reasonable correspondence of these values with AiX values found from the titration experiment is an additional argument for the validity of the simple binding model for protein interaction with denaturants. (e) Depression of the enthalpy and entropy of protein denaturation in the presence of denaturants Additional binding of the denaturant upon protein unfolding should be associated with the enthalpy and entropy effects, which can be calculated using A@ and AB of the binding: SAiH*

= (n”-nN)A@(a),

(21)

6A#*

= (n” -nN)As^(a),

(22)

Ad (J mol-‘) - 420 -1060 - 1670 -2310 -2990 -3710 -4400

where AH(a) and A&a) are determined by equations (18) and (19). The corresponding values are given in Table 4. The same Table also gives the values esti-, mated from the scanning calorimetric experiments by extrapolation from the transition temperature T, to 25°C using equations: A;H(T)

= A,UH(TJ-A;C,(T,-T),

A$S(T) = A:W”J T t

-A#?,

x ln(T,/T).

(23) (24)

The correspondence of these two sets of data obtained from the scanning calorimetric and the calorimetric titration experiments is an additional argument for the validity of the found thermodynamic characteristics of binding of GdmCl and urea with proteins, i.e. the argument for the efficiency of a simple binding model with the independent binding sites in describing protein interaction with these denaturants. Under the simple binding we certainly assume competitive binding with water (Tanford, 1970). (f) Correlation

din

mol-‘)

-43 -9.3 - 125 - 14.6 -159 -162 -15.9

- 1.7 -38 -54 -6.7 -7.7 -8.6 -9.1 --

0.155 0.347 0491 0.606 0.702 0.777 0831

Ad (J K-’

of the number of binding sites with structural parameters

To understand the mechanism of denaturant binding it is necessary, first of all, to find out what structural parameters of the protein molecule determine the number of binding sites of the given denaturant. Table 8 presents various structural characteristics of the examined proteins in the folded (native) and unfolded states. These include the total surface area of all polar and non-polar groups, the number of polar groups and the number of proton acceptor and donor groups. At the end of the Table are given the averaged values of these parameters calculated per one binding site and the correlation coefficients. In the case of urea, the best correlation is observed between the number of binding sites and the total number of exposed polar groups. It appears as if one urea molecule is bound by two hydrogen-bond-forming groups of the protein. For GdmCl we have almost the same situation, the best correlation is observed between the number of binding sites and the number of polar groups, but here it appears as if one binding site is formed by four or five hydrogen-bond-forming groups. It also

502

G. I. Makhutadze

and P. L. Privalov

Table 8 Correlation

of number

of denaturant

binding

sites with structural

Cytochrome Structural

or binding parameters

c

parameters

Ribonuclease

of proteins

Lysozyme

N

I’

x

u

N

u

Number of bind sites for urea Number of bind sites for GdmCl Number of amino acid residues Total ASAg Non-polar ASAQ: Polar ASA Peptide unit ASAS Number of hydrogen bonding groups Number of hydrogen acceptor groups Sumber of hydrogen donor groups

48 37 104 6062 3139 2797 1241 170 82 88

142 56 104 17,856 9326 7914 4674 303 147 156

122 43 124 6769 3395 3374 1366 212 106 106

240 74 124 20,094 9758 10,336 5700 386 193 193

119 57 129 6694 2690 3996 1685 229 101 128

222 82 129 20,784 9969 10.815 6285 411 182 229

Size of binding site for urea in terms of: Number of amino acid residues Total ASAg Non-polar ASA§ Polar ASAG Peptide unit ASAS Number of hydrogen bonding groups Number of hydrogen acceptor groups Sumber of hydrogen donor groups

22 126.3 65.4 58.3 259 3.5 1.7 1.8

97 125.7 65.7 5%57 32.9 2.1 I.0 I.1

0.5 83.7 40.7 43.1 23% 1.6 0.8 0.8

1.1 563 22.6 336 14.2 1.9 0.8 1.1

66 908 435 47.2 27.4 1% 0.X 1.0

0.8 82.4 40.1 41.5 21.9 1.9 0.9 I.0

025 085 0.80 092 089 095 096 0.9 I

Size of binding site for GdmCl in terms of: Number of amino acid residues Total ASA$ Non-polar ASAP Polar ASA I’eptide unit ASA§ Number of hydrogen bonding groups Number of hydrogen acceptor groups Number of hydrogen donor groups

2.8 163.X 848 756 33.5 46 2.2 24

1.9 318.9 166.5 141.3 83.5 ;?4 2.6 28

I.7 271.5 131.9 1397 77.0 52 26 2%

23 117.4 47.2 70.1 296 4.0 1.8 22

I.6 253.5 121.6 131.9 76.6 .50 2.2 r&8

2.1 223.7 109.2 112.3 59-i $9 2.3 N.i

-031 0.8 1 0.74 090 987 0.94 0.86 0.97

1.0 5.5..5 27.8 27.7 Il.2 1.7 0.9 0.9 2.9 157.4 790 78.5

31.8 4.9 2,.5 2.5

Meant

m

t Does not include the data for cytochrome e in the nat,ive state due to unclear data for the heme group $ Correlation coefficient. 0 Water accessible surface area(s) (ASA) were taken from Privalov $ Makhatadze (1990).

appears that the proton acceptor groups have some advantage in binding denaturants, especially a GdmCl molecule, which is likely to be bound by two proton acceptor groups. This is what one could expect, taking into account that all four groups of the GdmCl molecule that are able to form hydrogen bonds are weak proton donors and, of the five groups of urea able to form hydrogen bonds, four are proton donors and only one is a proton acceptor. The correlation of the estimated number of binding sites with the exposed non-polar surface area is rather poor. Therefore, one can hardly assume that urea and GdmCl interact with nonpolar groups of protein. This does not mean, however, that they cannot influence the protein stability by changing the hydrophobic interactions, as was supposed by many authors (see e.g. Breslow & Guo, 1990; Creighton, 1991). Denaturants in high concentration could affect properties of water determining hydrophobic interactions (Rupley, 1964). However, this indirect influence of denaturants through water cannot be tested by the protein titration experiment. Jt should be noted that our conclusion that urea and GudmCl are interacting mainly with polar groups of protein, and that these interactions are polyfunctional, is in accord with the earlier finding

of Robinson & Jencks (1965). Studying the solubilit,ies of compounds modeling the peptide groups in aqueous solutions of urea and GdmCl, they concluded that there is polyfunctional hydrogen bonding between the peptide groups and denat,urant molecules and that the interactions with non-polar groups are not dominant. The fact that the total number of bound molecules of urea and GdmCl correlates with the number of peptide groups in proteins has also been shown by Lee and Timasheff (1974) and Prakash et al. (1981) studying the volume effects of protein interaction with urea and GdmCl. (g) The role of hydrogen The large enthalpy indicates

that

enthalpic

of binding forces

bonding of urea and GdmC1

are involved

in their

binding. Therefore, the calorimetrically observed binding cannot be caused by electrostatic or hydrophobic interactions, but mainly by hydrogen bonding. Judging by the magnitude of the enthalpic effect, there should be multiple hydrogen binding. This assumption is confirmed by correlation of the number of binding sites with st,ructural parameters considered above, which showed that the binding site is likely to be formed by several polar groups. A

Protein

Interaction

503

with Denaturants

900 800 700 3000 T z E

600 2500 5cxl

z ‘2

2000

400

I I500

300 200 100 0 Molarlty

or actlvlty

(a)

Figure 8. The plot of A& against the concentration multiple hydrogen bonding of urea with peptide groups was recently revealed by crystallographic studies of diketopiperazine crystals (Thayer et al., 1992). A multiple binding by hydrogen bonds explains why GdmCl, which is a strong electrolyte, figures as a single molecule in binding (Pfeil et al., 1991) and why the pH of the solution does not have much influence on its binding to proteins. From the intrinsic binding characteristics obtained, it is clear that the action of the enthalpic force in binding is largely abolished by the negative entropy effect. This entropic effect reflects, evidently. the immobilization of a denaturant molecule on the protein surface. But it also might be partly caused by the fixation of protein groups at some configuration if the binding sites for urea and GdmCl are formed by several polar groups. This should result in a decrease in flexibility of the polypeptide chain, an increase in its rigidity, and an expansion of its hydrodynamic volume. Moreover, it should lead to some asymmetry in the conformation of the polypeptide chain that should be reflected in its circular dichroism spectrum. This effect of urea binding was proposed many years ago by Tiffany & Krimm (1972, 1973) but the idea was ignored, perhaps because it threatened the comforting point of view that a protein in concentrated solutions of urea and GdmCl can be regarded as a standard of a completely unfolded polypeptide chain in a random coiled conformation. Our study of the conformation of proteins in a denatured state (Privalov et al., 1989) raised new doubts of the correctness of this point of view and forced us to undertake this study of the thermodynamics of protein interaction with denaturants. The results obtained definitely show that the protein state in concentrated urea and GdmCl solution is not as simple as it is usually assumed to be and, without special analysis, can

(b)

(0) and activity

(0) of (a) GdmCl and (b) urea.

hardly be used as a standard state in considering protein unfolding/refolding process.

a

(h) Influence of denaturants on protein stability Let us now consider the frequently discussed question as to whether the thermodynamic parameters characterizing protein stability, particularly the Gibbs free energy of protein unfolding, are proportional to the denaturant concentration or to its activity (see e.g. Pace, 1975, 1986, 1990; Schellman, 1987a; Pace et aE., 1990). This is an important problem because a linear extrapolation is widely used for determining the protein stability in aqueous solutions without denaturants from the data that are obtained in the presence of denaturants. If the protein interaction with urea and GdmCl could be considered as a weak selective interaction then one would expect that the Gibbs energy of protein unfolding would be proportional to the denaturant’s concentration (Schellman, 1987aJ). However, if protein interaction with these denaturants can be regarded as a simple binding, the total Gibbs energy of binding, and thus the Gibbs energy of protein unfolding, would not be a linear function of the denaturant concentration or activity (see eqn (13)). The deviation from linearity is rather small, especially in the case of urea (Fig. 8): In the case of GdmCl this deviation amounts to about 10 to 15% at zero concentration of denaturant. This might be the reason that the unfolding Gibbs energy values for the metmyoglobins from the various sources obtained by the linear extrapolation to zero concentration of GdmCl were found to be in poor correspondence with the values determined by direct calorimetric measurements of the denaturation heat effects (Kelly & Holladay, 1990). Yao & Bolen (1992) recently came

504

G. I. Makhatadze

to the same conclusion. They studied protein denaturation by urea and GdmCl and found that the Gibbs energy extrapolation to zero concentration of these two denaturants gives different values because of non-linear dependence for GdmCl. If the total Gibbs energy of protein interaction with denaturants is not a linear function of concentration, then one should expect that the parameter m = dAiG/dQ, which is the measure of the Gibbs energy dependence on the denaturant concentration, should not be a constant, but should decrease upon increasing the range of denaturant concentration in which protein denatures, i.e. it should decrease with increasing protein stability. In particular, this should take place when,,protein stability is changed by pH variation. It looks as if this is just what has been observed by Pace et al. (1990) in their studies of urea and GdmCl-induced denaturation of two different ribonucleases in solution at different pH values. (i) Concluding

remarks

Perhaps the most surprising conclusion that follows from the results considered above is that the simple binding model describes rather well all experimentally observed thermodynamic properties of proteins in the presence of urea and GdmCl, and has the ability to predict these properties for other proteins with known structure. This is surprising because the applicability of a simple binding model to the protein interaction with these denaturants in concentrated solution has been questioned seriously (Schellman, 1987a,b, 1990). The simple binding model probably works, because what is observed in calorimetric experiments is not just the presence of denaturant molecules in the vicinity of protein, the “weak selective interaction” that was considered by Schellman (1990). The large enthalpy of interaction shows that it is provided by forces that hardly can be regarded as weak, although their action is almost abolished by the large negative entropy effect of immobilization of denaturant molecules. It should be noted, however, that the fitting of some model to the experimental observations does not mean unconditionally that the considered model is the only possible model. Therefore, we are looking forward to the other, perhaps more elaborate, interpretations of the results presented above. We thank Dr W. F. Harrington and Dr J. A. Schellman for the helpful discussions of the manuscript in its preparation. We also acknowledge the support of a visiting faculty fellowship from the Institute of Biophysical Research on Macromolecular Assemblies which is funded in part by the NSF Biological Research Centers Award (DIR-8721059). References Arakawa, T. $ Timasheff, S. N. (1984). Protein stabilization and destabilization by guenidinium salts. Biochemistry, 23, 5924-5929.

and P. L. Privalov Breslow, R. t Guo, T. (1990). Surface-tension measurements show that chaotropic salting in denaturants are not just water-structure breakers. Proc. Nat. Acad. Sci., U.S.A. 87, 167-169. Creighton, T. E. (1991). Stability of folded conformations. Curr. Opin. Struct. Biol. 1, 5-16. Gill, S. J., Hutson, J., Clopton, J. R. & Downing, M. ( 196 1). Solubility of diketopiperazine in aqueous solutions of urea. J. Phys. Chem. 65, 1432-1435. Hedwig, G. R., Lilley, T. H. t Linsdell, H. (1991). Calorimetric and volumetric studies of the interactions of some amides in water and 6 mol dm-’ aqueous guanidinum chloride. J. Chem. Sot. Faraday Trans. 87, 2975-2982. Kelly, L. & Holladay, L. A. (1990). A comparative study of the unfolding thermodynamics of vertebrate metmyoglobins. Biochemistry, 29, 5062-5069. Kresheck, G. C. & Benjamin, L. (1964). Calorimetric studies of the hydrophobic nature of several protein constituents and ovalbumin in water and aqueous urea. J. Phys. Chem. 68, 2476-2486. Lee, J. C. & Timasheff, S. N. (1974). Partial specific volumes and interactions with solvent components of proteins in guanidine hydrochloride. Biochemistry, 13, 257-265. Pace, C. N. (1975). Stability of globular proteins. CRC Crit. Rev. Biochem. 3, 143. Pace, C. N. (1986). Determination and analysis of urea and guanidine hydrochloride denaturation curves. Methods Enzymd. 131, 266-280. Pace, C. N. (1990). Conformational stability of globular proteins. Trends B&h-em. Sci. 15, 14-17. Pace, C. N., Laurents, D. V. & Thompson, J. A. (1990). pH dependence of the urea and guanidine hydrochloride denaturation of ribonuclease A and ribonuclease Tl. Biochemistry, 29, 2564-2572. Pfeil, W. & Privalov, P. L. (1976a). Thermodynamic investigations of proteins. I. Standard functions for proteins with lysozyme as an example. Biophys. Chem. 4, 23-32. Pfeil, W. & Privalov, P. L. (19763). Thermodynamic investigations of proteins. II. Calorimetric study of lysozyme denaturation by guanidine hydrochloride. Biophys. Chem. 4, 33-40. Pfeil, W., Welfle, K. C Bychkova, V. E. (1991). Guanidine hydrochloride titration of the unfolded apocytochrome C studied by calorimetry. Studia Biuphysicu, 40, 5-12. Prakash, V.? Loucheux, C., Scheufele, S., Gorbunoff, M. I. & Timasheff, S. N. (1981). Interactions of proteins with solvent components in 8 M-UN%. Arch. B&hem. Biophys. 210, 455-464. Privalov, P. L. (1979). Stability of proteins. I. Small globular proteins. Advan. Protein Chem. 33, 167-241. Privalov, P. L. & Khechinaahvili, N. N. (1974). A thermodynamic approach to the problem of stabilization of globular protein structure: a calorimetric study. J. Mol. Biol. 86, 665-684. Privalov, P. L. & Makhatadze, G. I. (1990). Heat capacity of proteins. II. Partial molar heat capacity of the unfolded polypeptide chain of proteins: protein unfolding effects. J. Mol. Biol. 213, 385-391. Privalov, P. L. & Potekhin, S. A. (1986). Scanning microcalorimetry in studying temperature induced changes in proteins. Methods Enzymol. 131, 4-51. Privalov, P. L., Ptitayn, 0. B. & Birshtein, T. M. (1969). Determination of the stability of DNA double helix in an aqueous media. Biopolymers, 8, 559-57 1. Privalov, P. L., Tiktopulo, E. I., Venyaminov, S. Yu.,

Protein Interaction Griko, Yu. V., Makhatadze, G. I. & Khechinashvili. N. N. (1989). Heat capacity and conformation of proteins in the denatured state. J. Mol. Biol. 205, 737-750. Robinson, D. R. & Jencks, W. P. (1965). The effect of compounds on the urea-guanidinium class on the activity coefficient of acetyltetraglycine ethyl ester and related compounds. J. Amer. Chem. Sot. 87, 2462-2470. Rupley, J. A. (1964). The effect of urea and amides upon water structure. J. Phys. Chem. 68, 2002-2003. Schellman, J. A. (1955). The stability of hydrogen-bonded peptide structures in aqueous solution. Compt. Rend Trav. Lab. Carlsberg, 29, 230-259. Schellman, J. A. (1975). Macromolecular binding. Biopolyme~s, 14, 999-1018. Schellman, J. A. (1976). The effect of binding on the melting temperature of biopolymers. Biopolymers, 15, 999-1000. Schellman, J. A. (1978). Solvent denaturation. Biopolymers, 17, 1305-1322. Schellman, J. A. (1987a). Selective binding and solvent denaturation. Biopolymers, 26, 549-559. Schellman, J. A. (19873). The thermodynamic stability of proteins. Annu. Rev. Biophys. Biophys. Chem. 16, 115-137. Schellmen, J. A. (1990). A simple model for solvation in mixed solutions. Biophys. Chem. 37, 121-140. Schonert, H. & Stroth, L. (1981). Thermodynamic interaction between urea and the peptide group in aqueous solutions at 25 “C. Biopolymers, 20, 817-831.

with Den&wants

505

Sijpkes, A. H., Van De Kleut, G. J. & Gill, S. C. (1992). Urea-diketopiperazine interactions: a model for urea induced denaturation of proteins. Nature (London), in the press. Stokes, R. H. (1967). Thermodynamics of aqueous urea solutions. Aust. J. Chem. 20, 2087-2100. Tanford, C. (1968). Protein denaturation. Part A and B. Advan. Protein Chem. 23, 121-282. Tanford, C. (1970). Protein denaturation. Part C. Advan. Protein Chem. 24, l-95. Thayer, M. M., Hailtwanger, R. C., Allured, V. S., Gill, S. C. 6 Gill, S. J. (1992). Peptide-urea interactions as observed in diketopiperazine-urea cocrystal. Nature (London), in the press. Tiffany, M. K. & Krimm, S. (1972). Effect of temperature on the circular dichroism spectra of polypeptides in the extended state. Biopolymers, 11, 2309-2316. Tiffany, M. L. & Krimm, S. (1973). Extended conformations of polypeptides and proteins in urea and guanidine hydrochloride. Biopolymera, 12, 575-587. Wyman, J. & Gill, S. J. (1990). Binding and linkage. Functional chemistry of biological macromolecules. University Science Book, Mill Valley, CA. Yao, M. & Bolen, D. W. (1992). Evaluation of the adequacy of the linear extrapolation method for determining unfolding free energy changes. ASBMBI B&physical Society Joint Meeting, Houston, Texas (Abstract 1986).

Edited by P. von Hippel

Protein interactions with urea and guanidinium chloride. A calorimetric study.

The interaction of urea and guanidinium chloride with proteins has been studied calorimetrically by titrating protein solutions with denaturants at va...
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